Aberrant Hedgehog (Hh) signaling promotes brain, skin, prostate, endometrial, and gastric tract cancers. Amplification of the Hh transcriptional regulators, the Gli proteins, or mutations in SPOP, the substrate receptor of the ubiquitin ligase CRLSPOP, are associated with the development of cancer. SPOP recruits substrates such as Gli3 to CRLSPOP through linear SPOP-binding (SB) motifs. Surprisingly, our preliminary data show that Gli3 contains many weak SB motifs. SPOP has two oligomerization domains that together facilitate self-association into higher-order SPOP homo-oligomers, whose size depends on the SPOP concentration. Hence, Gli3 and SPOP are multivalent for each other, but the role of their high valency in regulating Gli3 ubiquitination is unclear. Interestingly, SPOP localizes t punctate structures in the nuclei, which we designate as nuclear SPOP bodies. These bodies can be detected by light microscopy and are likely facilitated by multivalent interactions. We hypothesize that (a) multivalent Gli3 and SPOP assemble into higher-order Gli3/SPOP complexes, which may form nuclear SPOP bodies in cells; and (b) multivalency generates ultrasensitivity of Gli3 recruitment and ubiquitination by CRLSPOP to protein concentration. Multivalency may be a general mechanism to regulate signaling but is poorly understood because of challenges inherent to the heterogeneous nature of higher-order complexes. We will use an innovative combination of biophysical, structural, biochemical, and cell biological techniques to: 1. Test the hypothesis that Gli3 and SPOP are highly multivalent by (a) determining the location and affinities of SB motifs in Gli3 and their sequence/affinity relationship; and (b) by elucidating how the two SPOP oligomerization domains synergize to promote higher-order SPOP homo-oligomers and how their valency depends on SPOP concentration. 2. Test the hypothesis that multivalency of Gli3 and SPOP functions in controlling ubiquitination by (a) determining the concentration-dependence of size and affinity of higher-order Gli3/SPOP complexes; and (b) by charting the ubiquitination efficiency towards Gli3 as a function of concentration and valency, and by determining the role of SPOP oligomerization and substrate binding for its localization in nuclear SPOP bodies. Improving our understanding of the regulation of Gli3 levels will provide important insight into Hedgehog signaling in health and disease. The proposed work will have significant impact for our understanding of newly identified cancer mutations in SPOP and for ubiquitous but understudied higher-order protein complexes.